Phosphoinositides (PIPns), e.g., phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are key signaling molecules in cellular communication. Specific PIPns recruit target signaling proteins and intracellular adapter proteins to inner membrane sites to activate protein kinases and thereby initiate signal transduction cascades to regulate endocytosis, protein sorting, cell migration, and apoptosis. Determining the ligand selectivity of a given PIPn binding protein has become an important issue in deciphering downstream effectors, and in implementing drug discovery efforts for potential therapeutics that could control protein-phosphoinositide interactions. We will develop two lipid overlay systems to identify PIPn-specific lipid recognition proteins (LRPs). Both methods will identify proteins bound to a membrane. First we will prepare PIPn-containing liposomes to detect surface-bound LRPs. Second, we will extend this methodology to use stable polymerized liposomes. Then, the two lipid overlay systems will be provided to collaborators and potential customers for evaluation; the optimum composition will be manufactured for sale. Finally, these new tools will be used to develop commercial products.